Composition of Strawberry Floral Volatiles and their Effects on Behavior of Strawberry Blossom Weevil, Anthonomus rubi
Raimondas Mozūraitis1 &David Hall2&Nina Trandem3&Baiba Ralle4&Kalle Tunström1&Lene Sigsgaard5&
Catherine Baroffio6&Michelle Fountain7&Jerry Cross2&Atle Wibe8&Anna-Karin Borg-Karlson9,10
Received: 15 May 2020 / Revised: 27 August 2020 / Accepted: 28 September 2020
#The Author(s) 2020
Abstract
The strawberry blossom weevil (SBW),Anthonomus rubi, is a major pest in strawberry fields throughout Europe. Traps baited with aggregation pheromone are used for pest monitoring. However, a more effective lure is needed. For a number of pests, it has been shown that the attractiveness of a pheromone can be enhanced by host plant volatiles. The goal of this study was to explore floral volatile blends of different strawberry species (Fragaria x ananassaandFragaria vesca) to identify compounds that might be used to improve the attractiveness of existing lures for SBW. Floral emissions ofF. x a.varieties Sonata, Beltran, Korona, and ofF. vesca, were collected by both solid-phase microextraction (SPME) and dynamic headspace sampling on Tenax. Analysis by gas chromatography/mass spectrometry showed the floral volatiles ofF. x ananassa.andF. vescawere dominated by aromatic compounds and terpenoids, with 4-methoxybenzaldehyde (p-anisaldehyde) andα-muurolene the major compounds produced by the two species, respectively. Multi-dimensional scaling analyses separated the blends of the two species and explained differ- ences betweenF. vescagenotypes and, to some degree, variation betweenF. x ananassavarieties In two-choice behavioral tests, SBW preferred odors of flowering strawberry plants to those of non-flowering plants, but weevils did not discriminate between odors fromF. x ananassaandF. vescaflowering plants. Adding blends of six synthetic flower volatiles to non-flowering plants of both species increased the preference of SBW for these over the plants alone. When added individually to non-flowering plants, none of the components increased the preference of SBW, indicating a synergistic effect. However, SBW responded to 1,4-dimethoxybenzene, a major component of volatiles fromF. viridis, previously found to synergize the attractiveness of the SBW aggregation pheromone in field studies.
Keywords Anthonomus rubi.Fragaria x ananassa.Fragaria vesca. Floral odors . Semiochemicals . Pest control
Introduction
The strawberry blossom weevil (SBW),Anthonomus rubi Herbst, (Coleoptera, Curculionidae) is an oligophagous spe- cies that feeds and reproduces on rosaceous plants (Popov
1996). Among its hosts are strawberry,Fragariaspp., rasp- berry,Rubus idaeusL., blackberry,Rubusspp. and rose,Rosa spp. (Hill1987; Popov1996). Early in spring, adults move to strawberry and raspberry from overwintering shelters, both inside the cropping area and from perimeter wild host plants
* Raimondas Mozūraitis [email protected]
1 Department of Zoology, Stockholm University, Stockholm, Sweden
2 Natural Resources Institute, University of Greenwich, Chatham Maritime, Kent ME4 4TB, UK
3 NIBIO, Norwegian Institute of Bioeconomy Research, NO-1431 Ås, Norway
4 Latvian Plant Protection Research Centre, Riga LV-1039, Latvia
5 Department of Plant and Environmental Sciences, University of Copenhagen, 1871 Frederiksberg C, Denmark
6 Agroscope, Research Center Conthey, 1964 Conthey, Switzerland
7 NIAB EMR, East Malling, Kent ME19 6BJ, UK
8 Norwegian Centre for Organic Agriculture, NO-6630 Tingvoll, Norway
9 Department of Chemistry, School of Engineering Science in Chemistry, Biotechnology and Health, KTH, Royal Institute of Technology, 10044 Stockholm, Sweden
10 Department of Chemical Engineering, Mid Sweden University, 85170 Sundsvall, Sweden
https://doi.org/10.1007/s10886-020-01221-2
/ Published online: 8 October 2020
(Alford1984). There, they feed on foliage, flower buds and open flowers. The female weevil usually oviposits a single egg in an unopened bud (Aasen et al. 2004; Jary 1931;
Leska1965) before severing the bud petiole, either partially or completely, preventing further development of the bud. The oviposition period lasts 1–2 months, during which more than 150 buds can be destroyed per female (Easterbrook et al.
2003). The larva develops and pupates inside the withered bud and emerges in late summer. After emergence, the young adult feeds on foliage for a few weeks before moving to an overwintering site (Hill1987).
The weevil is a serious pest of cultivated strawberry (Fragaria x ananassa) throughout Europe (Cross et al.
2001), causing bud damage to 5–90% of the crop ( A a s e n a n d T r a n d e m 2 0 0 6; C r o s s e t a l . 2 0 0 1; Labanowska 2004; Leska 1965; Kovanci et al. 2005;
Krauß et al. 2014; Popov 1995; Svensson 2002).
Resistance to pyrethroid insecticides makes effective con- trol of SBW challenging (Aasen and Trandem2006).
For many insects, pheromones are key cues in mate finding (Yew and Chung2015), and can play an impor- tant role in integrated pest control programs (Suckling et al. 2014). The male-produced aggregation pheromone of SBW was identified as a blend of grandlure I, grandlure II and lavandulol in a 1:4:1 ratio by Innocenzi et al. (2001). This pheromone blend is attractive to both sexes and is used as a lure in commercial monitoring of SBW (Cross et al.2006a, b). However, recent data sug- gested that, in order to control SBW populations, more effective lure formulations are needed (Baroffio et al.
2018). For a number of pests, the attractiveness of a pher- omone can be enhanced by host plant volatiles (Reddy and Guerrero2004). This has been demonstrated for other weevils of the genusAnthonomus(Dickens 1989; Muniz- Merino et al.2014). Furthermore, it has been shown that combining a floral volatile, 1,4-dimethoxybenzene, with the aggregation pheromone of A. rubi, gives improved trap catches (Wibe et al. 2014). This compound is now included in commercially available lures.
The objective of this study was to identify other host- plant compound(s) that might be combined with the SBW aggregation pheromone to improve attraction of SBW.
We aimed: (i) to determine whether SBW is able to dis- criminate between volatiles from flowering versus non- flowering strawberry plants of two species, Fragaria x ananassa and F. vesca; (ii) to determine whether SBW prefers volatiles of floweringF. x ananassaover those of flowering F. vesca; (iii) to identify components of the volatile floral blends of F. x ananassa and F. vesca;
and (iv) to determine whether selected components of these floral volatiles elicit behavioral responses by SBW in a laboratory bioassay when tested individually or in blends.
Methods and Materials
Insects and PlantsAdult SBW were collected from strawberry fields in SE Norway (N59.66, E10.69) in mid-May and transported to the laboratory at the Royal Institute of Technology, Stockholm. Both sexes were kept together on potted plants of non-flowering Fragaria x ananassa Duchesne (Rosales:
Rosaceae), variety Sonata, enclosed in 400 cm3plastic cups covered with nylon mesh. A moistened piece of cotton at the bottom of the cup served as a water source. The day before experiments, weevils were sexed and kept individually in plastic cups without plants. Sex was determined by the pres- ence of a thorn on each intermediary coxa of male weevils (Innocenzi et al.2002).
Fragaria x ananassaplants of varieties Sonata, Beltran and Korona were obtained from the Plantagen Sweden stores.
Wild strawberry, Fragaria vesca L. (Rosales: Rosaceae) plants were obtained from the strawberry genotype collection held at the Ecology Department, Swedish Agricultural University, Uppsala, Sweden. Location of the nine genotypes (I-IX) was the following: I–N59o59.287, E17o25.578; II– N59o54.932, E17o08.646; III–N59o54.632, E17o22.775; IV –N59o53.858, E17o29.500; V–N59o45.729, E17o20.524; VI –N59o40.672, E17o17.660; VII–N59o31.674, E17o19.662;
VIII – N59o21.411, E18o17.118; IX – N59o21.412, E18o17.118. The plants were kept under laboratory condi- tions: 16:8 h L:D photoperiod and ~ 18 °C. A 1000 W day- light lamp (type DRF, for use in greenhouses) was used as the light source.
Chemicals
B e n z a l d e h y d e ( > 9 8 % c h e m i c a l p u r i t y ) , 4 - methoxybenzaldehyde (p-anisaldehyde; > 98% chemical pu- rity), methyl salicylate (> 98% chemical purity) and benzyl alcohol (> 99% chemical purity) were purchased from Alfa Aesar (Ward Hill, Massachusetts, USA). (±)-Limonene (>
98% chemical purity), decanal (> 98% chemical purity), 1,4- dimethoxybenzene (> 99% chemical purity) and pentadecane (> 99% chemical purity) were obtained from Sigma-Aldrich AB (Stockholm, Sweden), whileα-muurolene (95% chemical purity) and analytical standards in Table1were available from the Ecological Chemistry group (Stockholm, Sweden).
Diethyl ether (redistilled, 99.9%) and cyclohexane (99.9%) were purchased from Carlo Erba Reagents (Val de Reuil, France).
Sampling and Analysis of Floral Volatiles
We used solid-phase microextraction (SPME; Rout et al.
2012) to sample volatiles of three varieties ofF. x ananassa
Table 1 Composition of odor blends from flowers ofFragaria ananasacultivars Sonata, Beltran and Corona andF. vescacollected by solid phase microextraction, and previously reported olfactory activity ofAnthonomus rubito the compounds
Mean TIC count/g dry weight/h ± SE (× 10 million)e
No. Of Compound GRa RIb IDc F. a.Sonata F. a.Beltran F. a.Corona F. vesca OAf
1 α-Pinene ΜΤ 1017 RC 1 ± 1 bg 3 ± 1 b 0 99 ± 19 a ‡
2 β-Pinene ΜΤ 1105 RC 0 tr 0 34 ± 23 ‡
3 3-Carene ΜΤ 1151 RC 1 ± 1 b 2 ± 1 b 0 18 ± 7 a ‡
4 Limonene ΜΤ 1196 RC 19 ± 6 b 19 ± 3 b 33 ± 8 b 159 ± 27 a §
5 β-Phellandrene MT 1207 L, RI tr tr 2 ± 2 a 9 ± 4 a §
6 (Z)-β-Ocimene MT 1239 RC 0 0 0 tr §
7 (E)-β-Ocimene MT 1249 RC 0 9 ± 9 a 0 24 ± 14 a §
8 p-Cymene ARMT 1260 RC tr tr tr 8 ± 5 a
9 Hexyl acetate E 1269 RC tr 2 ± 1 b 1 ± 1 b 10 ± 3 a ‡
10 1-Ethyl-2-methyl-benzene AR 1271 RC tr 2 ± 1 b 0 25 ± 5 a
11 Octanal AL 1283 RC 9 ± 1 c 18 ± 4 bc 38 ± 17 b 108 ± 34 a
12 (Z)-3-Hexen-1-yl acetate E 1312 RC 43 ± 30 ab 12 ± 4 b 53 ± 15 a 80 ± 12 a ‡
13 Methoxybenzene AR 1330 RC 10 ± 4 ab 2 ± 1 b 11 ± 3 a 0
14 6-Methyl-5-hepten-2-one K 1331 RC 5 ± 1 c 12 ± 3b 31 ± 12 b 86 ± 11 a
15 1-Hexanol OH 1354 RC 7 ± 3 ab 1 ± 1 b 14 ± 10 ab 46 ± 26 a ‡
16 methoxymethyl-Benzene AR 1379 RC 26 ± 19 a 2 ± 1 a 0 0
17 (Z)-3-Hexen-1-ol OH 1380 RC tr 1 ± 1 b 10 ± 6 a 27 ± 7 a ‡
18 Nonanal AL 1388 RC 42 ± 5 c 80 ± 10 b 212 ± 39 a 428 ± 80 a
19 Copaene ST 1483 RC 0 0 0 22 ± 14
20 Decanal AL 1493 RC 64 ± 10 b 171 ± 32 ab 310 ± 50 a 525 ± 134 a
21 Benzaldehyde AR 1501 RC 78 ± 4 ab 141 ± 55 a 40 ± 15 b 64 ± 10 ab
22 Linalool OMT 1534 RC tr 0 0 0 ‡
23 β-Caryophyllene ST 1587 RC 0 0 0 25 ± 18 §
24 Methyl benzoate AR 1602 RC 13 ± 2 a 9 ± 5 a 8 ± 1 a 12 ± 6 a §
25 3,6,6-Trimethyl-2-norpinanone OMT 1618 L, RI 0 0 0 11 ± 6
26 Acetophenone AR 1630 RC tr 3 ± 1 a 1 ± 1 a 13 ± 13 a
27 1-Ethenyl-4-methoxy-benzene AR 1661 RC 3 ± 2 b 26 ± 8 a 0 0
28 3-Ethyl-benzaldehyde AR 1690 RC 8 ± 3 b 5 ± 2 b 6 ± 2 b 24 ± 5 a
29 Germacrene D ST 1696 RC 0 0 0 2 ± 1 §
30 α-Muurolene ST 1716 RC 11 ± 2 b 10 ± 5 b 14 ± 6 b 604 ± 104 a
31 4-Ethyl-benzaldehyde AR 1718 RC 6 ± 2 b 10 ± 3 ab 6 ± 2 b 20 ± 5 a
32 (E,E)-α-Farnesene ST 1743 RC 0 2 ± 2 b 0 128 ± 34 a
33 Unidentified 1 (sesquiterpene) ST 1746 L, RI tr 2 ± 2 a 0 4 ± 3 a
34 Methyl salicylate AR 1754 RC 96 ± 18 b 59 ± 27 b 35 ± 18 b 268 ± 32 a §
35 TMTTd HT 1801 RC 0 0 0 15 ± 8
36 Dihydroα-ionone TK 1802 RC tr 12 ± 5 0 0
37 Unidentified 2 (sesquiterpene) ST 1847 L, RI 0 4 ± 2 a 12 ± 6 a 0
38 Benzyl alcohol CAS#: AR 1859 RC 310 ± 32 a 136 ± 63b 99 ± 24 b 312 ± 41 a
39 Benzyl isovalerate AR 1876 RC 4 ± 1 ab 22 ± 11 a 5 ± 3 ab 0
40 2-Phenylethanol AR 1894 RC 17 ± 2 ab 8 ± 3 b 88 ± 47 a 106 ± 48 a
41 1,4-Butanediol OH 1912 L, RI 0 0 0 20 ± 11
42 1,2-Benzisothiazole O 1931 L, RI 26 ± 14 a 6 ± 2 a 8 ± 1 a 14 ± 5 a
43 4-Metoxy-benzaldehyde ARE 1998 RC 1997 ± 541 a 1144 ± 433 a 1532 ± 536 a 0
44 Methyl 2-methoxy-benzoate ARE 2049 RC 12 ± 7 a 8 ± 4 a 10 ± 1 a 0
45 Benzyl 2-methyl-(EorZ)-2-butenoate ARE 2092 L, RI 30 ± 5 a 70 ± 46 a 23 ± 13 a 0
46 Hexahydrofarnesyl acetone TK 2120 RC 6 ± 2 b 19 ± 6 ab 14 ± 4 ab 35 ± 7 a
and flowers of different genotypes ofF. vescausing the same fiber type, headspace volume, and temperature during sam- pling. An internal standard was used to check for saturation of fibers. This is a sensitive technique and provided quantitative data for statistical comparisons among varieties and species.
Even so, comparison of quantities of different compounds in the same sample is not possible without use of labelled stan- dards, because of different affinities of the fiber for com- pounds and different vapor pressures of compounds (SPME guidelines2020). Thus, dynamic headspace sampling with an internal standard was also used for more reliable quantifica- tion of compounds and release rates.
S P M E s a m p l i n g w a s c a r r i e d o u t w i t h polydimethylsiloxane-divinylbenzene-coated fibers (65μm; Supelco, Sigma-Aldrich group, PA, USA) (Vas and Vekey2004), desorbed at 250 °C for 2 min in a gas chromatograph (GC) injector prior to sampling. A single, ready-to-flower bud from a potted strawberry plant was placed in a glass chamber (30 cm3) through an opening at the bottom, along with a filter paper (1 cm2) treated with 100 ng of pentadecane per 10μl of cyclohexane as internal standard. The opening was carefully sealed with aluminum foil. After the bud had opened, the SPME fiber was intro- duced close to the flower through a second opening in the chamber. Sampling of volatiles was carried out from 08.00
until 18.00 h covering the main period of emission (Ceuppens et al. 2015). After collection, the fiber was re- moved and desorbed directly in the injector of the gas chromatograph/mass spectrometer (GC/MS). Volatiles were collected from individual flowers ofF. ananassava- riety Sonata (N= 3), F. ananassa variety Beltran (N= 4), F. ananassavariety Corona (N= 4), andF. vesca(N= 9).
Dynamic headspace sampling (Millar and Haynes1998) was carried out on individual flowers ofF. ananassavariety Sonata (N= 3) andF. vesca(N= 3). A single, ready-to-flower bud was placed in the type of glass jar used for SPME sam- pling. Charcoal-purified and humidified air (50 ml.min−1) was supplied by a diaphragm vacuum pump (NMP 830 KNDCB;
KNF Neuberger Inc., Freiburg, Germany), and pulled through a glass collection tube containing Tenax TA adsorbent (50 mg; 60/80 mesh; Sigma-Aldrich AB, Sweden) by a sec- ond pump. Volatiles were collected from 08.00–18.00 h as above. After sampling, traps were extracted with 250 μl of redistilled diethyl ether, and 20 ng of pentadecane in cyclo- hexane was added as internal standard. Samples were concen- trated under a gentle flow of nitrogen and analyzed on the same day as collected.
After sampling, flowers were detached from the peduncle, placed in a glass beaker and kept in a thermostat at 60 °C for 72 h for determination of dry weight. For both sampling Table 1 (continued)
Mean TIC count/g dry weight/h ± SE (× 10 million)e
No. Of Compound GRa RIb IDc F. a.Sonata F. a.Beltran F. a.Corona F. vesca OAf
47 2-Phenoxyethanol AR 2122 RC 3 ± 2 b tr 4 ± 1 a tr
48 Unidentified 3 2133 28 ± 3 a 7 ± 3 b 26 ± 8 a 0
49 Unidentified 4 E 2240 15 ± 5 a 8 ± 6 a 25 ± 9 a 59 ± 20 a
50 4-Methoxybenzyl ethanol AR 2257 RC 60 ± 14 a 24 ± 6 b 34 ± 9 ab 0
51 3,4-Dimethoxybenzaldehyde AR 2365 RC 7 ± 5 a 3 ± 1 b tr 0
52 Unidentified 5 2376 16 ± 6 a 7 ± 5 a 19 ± 6 a 52 ± 13 a
53 Unidentified 6 2480 19 ± 7 a 11 ± 7 a 32 ± 5 a 60 ± 24 a
54 Unidentified 7 2487 49 ± 18 a 9 ± 6 a 6 ± 2 a 0
55 Benzyl benzoate AR 2629 RC 46 ± 8 a 136 ± 80 a 19 ± 10 a 0
56 Unidentified 8 2690 11 ± 7 a 3 ± 3 a 12 ± 7 a 0
57 Unidentified 9 2851 tr tr 0 44 ± 14
aGR = group of chemical compound (MT monoterpene; ARMT aromatic monoterpene; E ester; AR aromatic; AL aldehyde; K ketone; OH alcohol;
OMT oxygenated monoterpene; ST sesquiterpene; HT homoterpene; TK terpene ketone; ARE aromatic ester; O other compound)
bRI = retention index (DB-Wax fused silica capillary column 30 m × 0.25 mm i.d., 0.25μm film thickness)
cID = identification source; RC = reference compound; RI = retention index; L = NIST and MassFinder3 libraries
dTMTT = (3E,7E)-4,8,12- Trimethyltrideca-1,3,5,7,11-tetraene
eTIC = total ion chromatogram; SE = standard error of mean; tr = trace; F. a. Sonata (N= 3), F. a. Beltran (N = 4), F. a. Corona (N = 4), and F. vesca (N = 9)
fOA = olfactory activity reported inA. rubi; § Bichão et al.2005a,‡Bichão et al.2005b
gThe means indicated by the same letter in each row are not different (nonparametric Conover-Iman test,P< 0.05)
techniques, volatiles were also collected from empty glass chambers.
Samples were analyzed using a Varian 3400 GC coupled to a Finnigan SSQ 7000 MS (Thermo-Fisher Scientific, USA). A DB-Wax fused silica capillary col- umn (30 m length, 0.25 mm id, 0.25 μm film thickness;
Supelco-Sigma-Aldrich group, USA) was used. The col- umn oven was programmed from 40 °C for 3 min, then at 4 °C.min−1to 200 °C, then at 10 °C.min−1to 230 °C, and held for 9 min. The split/splitless injector temperature was 225 °C and the splitless period lasted for 60 s. Helium was used as carrier gas with an inlet pressure of 70 kPa.
The transfer line temperature was 235 °C. Electron ioni- zation mass spectra were determined at 70 eV with an ion source at 150 °C. Chromatographic profiles of volatiles were compared and compounds in large amounts, relative to those in blank samples, identified by comparison of mass spectral data and retention indices with synthetic standards (Table 2), along with data from NIST version 2.0 mass spectral search program (National Institute of Standards and Technology, USA). Relative amounts of compounds were determined as areas under chromato- graphic peaks. Absolute amounts of compounds trapped by aeration were quantified by applying standard calibra- tion curves derived from pentadecane at 0.5 ng, 1 ng, 10 ng and 50 ng.
Behavioral Tests
Behavioral responses of SBW to natural and synthetic odors were tested in two-choice olfactometers. The test area was illuminated with a quartz metal halide lamp ( H P I - T P l u s 4 0 0 W ; P h i l i p s , A m s t e r d a m , t h e Netherlands) placed 180 cm above the olfactometers.
The olfactometers comprised three layers of acrylic plastic (each layer 0.5 cm thick) sandwiched together with an arena cut out in the middle layer and consisting of a cen- tral zone (2 × 2.5 cm) with two tapered arm zones (4 cm length from the air inlet to the central zone and 0.4 cm to 2.5 cm width at the inlet and the central zone, respective- ly) (Hambäck et al. 2003) (Fig. 5a). One vacuum dia- phragm pump delivered stimuli in purified and humidified air to the arms of the olfactometer and another pump was connected via Teflon tubes to the top so as to withdraw air at ca. 3 ml.sec−1. Four olfactometer trials could be run simultaneously, testing an odorant of the same type.
Before each trial, a weevil was allowed to acclimatize inside the olfactometer for 3 min. During delivery of the stimulus, the weevil’s position in the arena was noted at 30 s intervals for 15 min, giving a total of 30 recordings for each individual weevil. The responsiveness of a SBW was assessed by calculating the percentage of records in each arm. Each weevil was only used once in an
experiment, and weevils that were inactive in the central zone for more than 5 min during trials were excluded from the analysis. Between trials, the olfactometers were washed with water and a mild detergent.
In the first experiment, weevil preference to the olfactom- eter arms without olfactory cues was tested to confirm lack of bias in the olfactometer. In the second experiment, weevil responses to odors from flowering versus non-flowering strawberry plants were evaluated. For the non-flowering plants, buds or flowers were removed 2 d prior to the exper- iment. A single potted flowering and non-flowering F. x ananassaplant were placed separately in a polyester cooking bag (25 × 40 cm; Toppits, Minden, Germany), while 4–5 pot- ted flowering and non-floweringF. vescaplants (collected at N59o21.412, E18o17.118) of total leaf area approximately equal (estimated by visual evaluation) to that of the F. x ananassa plant were placed together in another polyester cooking bag. OneF. x a.var. Sonata flower corresponded to four F. vesca flowers, based on dry weight. The pots were covered with aluminum foil in order to minimize soil volatiles in the headspace. Purified and humidified air was delivered at a ca. 12 ml.sec−1from the bottom of the bag, and volatiles were collected at the top of the bag.
In the third experiment we tested preference for flowering F. vescaversus floweringF. x a.var. Sonata plants, using five wild strawberry plants with eleven flowers and one garden strawberry plant with three flowers, respectively.
In the fourth experiment, the responses of SBW to synthet- ic chemicals added to non-flowering plants were tested against plants alone. Compounds were dissolved in cyclohexane and 10μl of solution applied to 2 cm2of filter paper in a glass vial (10 cm3) positioned close to the plant. Single compounds were tested at a dose of 100 ng. Two six-component blends of synthetic compounds were made to represent floral bouquets of F. x ananassa var. Sonata (FAS) and F. vesca (FV), r e s p e c t i v e l y. T h e F A S b l e n d c o m p r i s e d 4 - methoxybenzaldehyde, benzaldehyde, benzyl alcohol, methyl salicylate, limonene and decanal at loadings of 200, 15, 51, 20, 9 and 40 ng, respectively. The FV blend comprised α- muurolene, benzaldehyde, benzyl alcohol, methyl salicylate, limonene and decanal at loadings of 30, 25, 45, 60, 70 and 80 ng, respectively. The loading ratios and amounts of syn- thetic odorants were selected to emit profiles similar to those of strawberry flowers. Five components in these two synthetic blends were common, consistent with the observation that SBW showed no preference for flowering plants of either species. All five compounds were previously shown to elicit electrophysiological responses from the antennae of SBW by Bichão et al. (2005a). Furthermore, these compounds repre- sent different classes of floral volatiles and would be econom- ically feasible for practical control use. In addition, the FAS blend included 4-methoxybenzaldehyde, the major compo- nent in floral volatiles from cultivated strawberry, while the
Table 2 Composition of volatiles from flowers ofFragaria ananasacultivar Sonata andF. vescaflowers collected by dynamic headspace technique Mean rate ± SE (ng/g dry weight/h)e
No Compound GRa RIb IDc F. a. Sonata F. vesca
1 α-Pinene MT 1017 RC 0.5 ± 0.29 4.4 ± 3.07deff
Hexanal AL 1076 RC 0.3 ± 0.15 0
2 β-Pinene ΜΤ 1105 RC 0.2 ± 0.06 0
3 3-Carene MT 1151 RC 0.3 ± 0.12 0
Heptanal AL 1180 RC 0.3 ± 0.02 2.4 ± 1.8f
4 Limonene MT 1196 RC 1.6 ± 0.52d 11.9 ± 1.46bcd
Propylbenzene AR 1198 RC 0.3 ± 0.20 0
5 β-Phellandrene ΜΤ 1207 L, RI 0.1 ± 0.04 0
(E)-2-Hexenal AL 1208 RC 0.1 ± 0.05 3.5 ± 2.44ef
6 (Z)-β-Ocimene MT 1239 RC tr 0
7 (E)-β-Ocimene MT 1249 RC tr 3.0 ± 1.2ef
8 p-Cymene ARMT 1260 RC tr 0
10 Hexyl acetate E 1269 RC 0.1 ± 0.06 0
11 Octanal AL 1284 RC 3.7 ± 0.60 cd 6.8 ± 0.45de
12 (Z)-3-Hexen-1-yl acetate E 1312 RC 2.1 ± 1.07d 9.1 ± 1.91bcd
13 Methoxybenzene AR 1331 RC 0.7 ± 0.55 0
14 6-Methyl-5-hepten-2-one K 1332 RC 9.7 ± 2.16 tr
15 1-Hexanol OH 1355 RC 2.1 ± 0.25 0
16 Methoxymethyl-benzene AR 1380 RC 0.3 ± 0.12 0
17 (Z)-3-Hexen-1-ol OH 1381 RC 0.8 ± 0.26 0
18 Nonanal AL 1389 RC 13.0 ± 1.63b 9.7 ± 0.71bcd
19 α-Copaene ST 1484 RC tr 0
20 Decanal AL 1493 RC 18.9 ± 7.78ab 8.9 ± 1.76bcd
21 Benzaldehyde AR 1501 RC 9.6 ± 0.76b 14.5 ± 2.58ab
22 Linalool OMT 1534 RC 0.2 ± 0.08 7.3 ± 1.71cde
23 β-Caryophyllene ST 1587 RC 0.0 2.5 ± 0.64f
Undecanal AL 1597 RC 1.5 ± 0.4d 0
24 Methyl benzoate AR 1602 RC 2.1 ± 0.41d 0
26 Acetophenone K 1630 RC 0.2 ± 0.14 8.2 ± 2.21bcd
27 1-Ethenyl-4-methoxybenzene AR 1661 RC 1.0 ± 0.11 0
29 Germacrene D ST 1696 RC 0.4 ± 0.27 1.4 ± 0.73f
Unidentified 1 (sesquiterpene) ST 1715 RC 0.1 ± 0.04 0
30 α-Muurolene ST 1716 RC 0.1 ± 0.05 18.5 ± 1.79a
32 (E,E)-α-Farnesene ST 1743 RC 1.2 ± 0.26d 0
34 Methyl salicylate AR 1754 RC 2.6 ± 0.41c 9.0 ± 1.47bcd
35 TMTTd HT 1801 RC 0.5 ± 0.22 4.1 ± 0.86ef
Unidentified 2 (sesquiterpene) OST 1847 L, RI 11.9 ± 7.55abc 0
38 Benzyl alcohol AR 1859 RC 11.0 ± 5.18ab 10.8 ± 2.35bc
39 Benzyl isovalerate AR 1875 RC tr 0
40 2-Phenylethanol AR 1893 RC 3.2 ± 2.79 cd 0
42 1,2-Benzisothiazole O 1930 L, RI tr 0
43 4-Methoxybenzaldehyde AR 1997 RC 23.0 ± 2.37a 0
44 Methyl 2-methoxybenzoate ARE 2048 RC tr 0
45 Benzyl 2-methyl-(EorZ)-2-butenoate ARE 2091 L,RI tr 0
(Z)-3-Hexen-1-ol benzoate E 2103 RC tr 0
46 Hexahydrofarnesyl acetone TK 2119 RC tr 0
Unidentified 3 2132 0.6 ± 0.45 0
FV blend containedα-muurolene, the major component in volatiles from wild strawberry.
In the fifth experiment, the responses of SBW to 4- methoxybenzaldehyde added to non-floweringF x ananassa var. Sonata plants at doses of 10 ng, 100 ng and 1000 ng were investigated.
Data Analysis
To monitor saturation of SPME fibers, the amounts of pentadecane adsorbed were compared by Mann Whitney U test, using Statistica software version 6.0.
To compare amounts of floral volatiles between F. x ananassa varieties and F. vesca, data were analyzed by Kruskal-Wallis a non-parametric test followed by a Conover-Iman test using R (version 4.0.2) and Rstudio (ver- sion 1.3.959).
To assess and visualize associations among odor blends of strawberry flowers sampled by SPME, a multidimensional scaling (MDS) analysis with a Bray-Curtis index was per- formed on absolute amounts, expressed as areas under chro- matographic peaks, using R (version 4.0.2) and Rstudio (ver- sion 1.3.959), with the metaMDS function in the vegan pack- age (version 2.5–6). The results were visualized using ggplot2 (version 3.3.2). Prior to analysis, the data were square root transformed. Amounts of volatiles were also used to show degree of similarity of odor bouquets betweenF. x ananassa varieties andF. vescaby cluster analysis, based on Euclidian distance using Statistica software version 6.0.
The behavioral responses of SBW in two-choice olfactom- eter tests were analyzed by nonparametric Wilcoxon matched- pairs signed-ranks test using the Statistica software version 6.0.
Results
Chemical Composition and Variation of Strawberry Floral Odor Blends
Using SPME, 46, 39 and 49 compounds were detected in floral volatiles fromF. x ananassavarieties Sonata, Beltran, and Korona, respectively. In the flower headspace ofF. vesca, 41 compounds were detected (Table1). Monitoring saturation of SPME fibers by adding 100 ng of pentadecane as internal standard to the samples revealed no differences in amount of pentadecane trapped on the fibers in blank samples (empty glass jars; median TIC count 17,934,247) versus F. vesca flowers (median TIC count 15,258,935) and versus flowers of F. x ananassa variety Sonata (median TIC count 11,565,433) (Mann Whitney U test, N= 5, P= 0.222 and N= 3,P= 0.071, respectively).
Using the dynamic headspace sampling technique, 49 com- pounds were detected in floral volatiles collected fromF. x ananassavariety Sonata and 18 compounds from F. vesca (Table2). For both techniques, all samples were from single flowers. GC/MS analyses revealed that floral volatile blends of both species were dominated by aromatic compounds and terpenoids. From the flowers of F. x ananassa, 4- methoxybenzaldehyde (p-anisaldehyde) was collected in the largest quantity, and from F. vesca, α-muurolene (Fig. 1, Tables1and2).
Multidimensional scaling (MDS) analysis, using data from SPME samplings, showed that odor blends released fromF. x ananassaandF. vescaflowers were separated from each other (Fig. 2). The first MDS axis explained separation between specimens ofF. x ananassaandF. vescaspecies. Twenty four compounds had significantly higher amounts per gram of dry flower weight released per hour for F. vescathan for F. x Table 2 (continued)
Mean rate ± SE (ng/g dry weight/h)e
No Compound GRa RIb IDc F. a. Sonata F. vesca
Unidentified 4 2239 0.1 ± 0.4 0
50 4-Methoxybenzyl alcohol AR 2256 RC tr 0
55 Benzyl benzoate AR 2629 RC 0.2 ± 0.12 0
aGR = group of chemical compound (MT = monoterpene; AL = aldehyde; AR = aromatic; ARMT = aromatic monoterpene; E = ester; HT = homoterpene; K = ketone; O = other compound; OH = alcohol; OMT = oxygenated monoterpene; ST = sesquiterpene; TK = terpene ketone
bRI = retention index (DB-Wax fused silica capillary column 30 m × 0.25 mm i.d., 0.25μm film thickness)
cID = identification source; RC = reference compound; RI = retention index; L = NIST and MassFinder3 libraries
dTMTT = (3E,7E)-4,8,12- Trimethyltrideca-1,3,5,7,11-tetraene
eSE = standard error of mean; tr = trace;F. a.Sonata (N = 3) andF. vesca(N = 3)
fThe means indicated by the same letter in each column are not different (nonparametric Conover-Iman test, P < 0.05, calculated for the compounds with amount exceeding 1 ng)
ananassa flower samples (Table 1). The odor blends of F. vescaflowers were characterized by seven unique com- pounds, including six terpenoids [α-copaene (19), β- caryophyllene (23), 3,6,6-trimethyl-2-norpinanone (25), germacrene D (29), (3E,7E)-4,8,12- rimethyltrideca- 1,3,5,7,11-tetraene (TMTT) (35) andβ-phellandrene (5)], as well as one alcohol [1,4-butanediol (41)]. Of the 24 com- pounds, 15 were terpenoids, three aromatics, three aldehydes, and one each of a ketone, ester and unidentified compound (Fig.2, Table1). Floral volatile blends fromF. x ananassa contained 17 unique compounds, including 10 aromatics, two terpenoids, one ketone and four unidentified compounds (Table1).
The second MDS axis explained differences between F. vescagenotypes and some of the variation betweenF. x ananassavarieties (Fig.2). A significant correlation (r =
0.5356,P= 0.017) (Fig.3) was found between geographical separation ofF. vescagenotypes and differences in floral odor blends, expressed as projection distances of blends on the second MDS axis in Fig.2. Flowers of variety Sonata had the odor blend most distinct from the other two varieties ofF. x ananassa(Fig.4), and were characterized by large a m o u n t s o f b e n z y l a l c o h o l ( 3 8 ) , 3 , 4 - dimethoxybenzaldehyde (51) and linalool (22). Odor blends of variety Korona were distinguished by larger amounts ofβ-phellandrene (5), (Z)-3-hexen-1-ol (17) and nonanal (18) and the absence of eight compounds compared to the other two cultivars (Table1). (E)-β-Ocimene (7), 1- ethenyl-4-methoxybenzene (27), (E,E)-α-farnesene (32), an unidentified sesquiterpene (37) andβ-pinene (29) were present in larger quantities in the odor blends of variety Beltran.
Intensity (arbitrary units. Number has to be multiplied by10 )6
F. a. var. Corona Total intensity 8.0 x 107 F. a. var. Sonata Total intensity 8.2 x 107
F. a. var. Beltran Total intensity 9.3 x 107
Time (min)
0 5 10 15 20 0 5 10 15 20
0 5 10 15 20
0 5 10 15
20 F. vesca
Total intensity 2.0 x 107
6 10 14 18 22 26 30 34 38 42 46 50 54
1 2 3
4 5 67 8
9 10 11
12 13
14 18 16
15 18
19
30
17 20
31
21 22
23 24
25 26
27
28 29 32
3334
35 36
37
38 39
40
41 42
44
45
46 48
48
49 50
51
52 43
38
43 47
53 54
55 56
57 IS 52
IS
Fig. 1 Total ion chromatograms from gas chromatography/mass spectrometry analyses of floral odors collected by solid phase microextraction headspace sam- pling of a single flower of Fragaria x ananassavarieties Sonata, Beltran, Korona, and F. vesca. (DB-Wax fused silica capillary column; numbered chromatographic peaks are listed in Tables1and2; IS pentadecane internal standard)
I
II III IV
V
VI VIIVIII
IX
- 0.50 - 0.25 0.00 0.25
-0.6 -0.3 0.0 0.3 0.6
First MDS axis
Species
F. x a. var. beltran F. x a. var.corona F. x a. var.sonata F. vesca
Second MDS axis
Fig. 2 Score plot of odor blends sampled by solid phase microextraction of headspace of single flowers ofFragaria x ananassavarieties Sonata, Beltran, Korona, andFragaria vescapotted plants. Roman numerals represent genotype of F. vescaplants; MDS = multidimensional scaling
Olfactory Preferences ofAnthonomus rubi
SBW showed no preference for either olfactometer arm when no olfactory cue was present, showing no inherent bias in the bioassay (Fig.5b). When given the choice between odors of floweringF. x ananassa var. Sonata plants and odors from non-flowering plants, both male and female SBW preferred those from the flowering plant (N= 16,Z= 2.811,P= 0.005 andN= 14,Z= 1.977, P= 0.048, respectively) (Fig. 5c). In further experiments, SBW were not separated by sex.
When testing SBW preference to odors of flowering versus non-flowering F. vesca, more weevils chose odors of flowering plants (N= 14,Z= 2.068,P= 0.041). No preference was observed between odors of floweringF. x a.var. Sonata andF. vescaplants (N= 11,Z= 0.044,P= 0.965) (Fig.5c).
SBW preferred non-floweringF. x a.var. Sonata strawber- r y pl an t s w i t h t he F A S s y n t h et i c o d or b l e n d ( 4 - methoxybenzaldehyde, benzaldehyde, benzyl alcohol, methyl salicylate, limonene and decanal) compared to non- flowering plants alone (N= 11, Z = 2.667, P= 0.008).
Similarly, SBW preferred non-floweringF. vescaplants with the FV blend (α-muurolene, benzaldehyde, benzyl alcohol, methyl salicylate, limonene and decanal) over flowering plants alone (N= 12,Z= 2.746,P= 0.006) (Fig.5d).
None of the compounds in the two blends when added individually at 100 ng to non-flowering strawberry plants in- creased or decreased preference of SBW over non-flowering plants alone (benzaldehydeN= 11, Z = 1.156,P= 0.248; ben- zyl alcohol N= 10, Z = 1.376, P= 0.169; methyl salicylate N= 13, Z = 0.069, P= 0.944; α-muurolene N= 14, Z = 0.549,P= 0.583; limoneneN= 6, Z = 1.531,P= 0.245, and decanalN= 7, Z = 1.726, P= 0.507) (Fig.5d). Data on the effects of limonene and decanal should be considered prelim- inary due to the low number of replicates.
In the final bioassay experiment, SBW did not discriminate between odors released from non-floweringF. x a.var. Sonata strawberry plants and those from non-flowering plants of the same variety with added 4-methoxybenzaldehyde at 10 ng or 100 ng doses (N= 16, Z = 1.172,P= 0.241 andN= 12, Z = 1.579,P= 0.114, respectively). When the dose was increased to 1000 ng, weevils preferred the side with the non-flowering plant alone (N= 10, Z = 2.803,P= 0.005) (Fig.5e).
Discussion
Chemical Composition and Variation of Strawberry Floral Odor Blends
The composition of floral volatile emissions from several spe- cies of strawberry have been reported previously, including Fragaria x ananassa(Bichão et al. 2005a; Ceuppens et al.
2015; Hamilton-Kemp et al.1990,1993; Klatt et al.2013), F. virginiana Duchesne (Ashman et al. 2005), F. vesca (Blažytė-Čereškienė et al. 2017; Wibe et al. 2014) and F. viridisDuchesne (Blažytė-Čereškienėet al.2017). In those studies, static and dynamic headspace collections as well as hydro-distillation techniques were used to sample floral com- pounds from cut and intact flowers, making it difficult to compare data. The amounts of volatiles produced by individ- ual strawberry flowers are very small; hence, we used SPME to sample volatile profiles under standardized conditions. This provided a highly sensitive technique able to detect more com- pounds than in the above studies, and also provided quantita- tive data for comparisons of varieties and species. Dynamic headspace sampling with an internal standard was also used for more reliable quantification of compounds and release rates.
Spatial distance (%)
Projection distance on PC2 axis (%)
r = 0.5356, P = 0.0017
0 20 40 60 80 100
0 20 40 60 80 100
Fig. 3 Relationship between geographic separations ofFragaria vesca genotypes in the field and differences in floral odor blends expressed as projection distances of blends on the second axis in the multidimensional scaling score plot in Fig.2. Both spatial and floral blend projection distances were transformed to percentage scale assigning the largest distance to 100%. The largest spatial distance between origins of two genotypes was 95 km
0 5.0 10.0 15.0 20.0
Linkage Distance F. vesca
F. x a. Corona F. x a. Beltran F. x a. Sonata
Fig. 4 Dendrogram of odor blends sampled by solid phase microextraction of headspace of single flowers ofFragaria x ananassa varieties Sonata, Beltran, Korona, andFragaria vesca. Dendrogram was obtained by cluster analysis based on Euclidian distance. Numbers on x axis have to be multiplied by 109
We found that 4-methoxybenzaldehyde (p-anisaldehyde) was the major constituent of the floral volatile blends released by all threeF. x ananassavarieties. This contrasts with previ- ous reports in which benzaldehyde was present in the largest amount in volatile emissions of F. x ananassa varieties Darselect, Honeoye (Klatt et al.2013) and Korona (Bichão et al.2005a). (E,E)-α-Farnesene and limonene were reported as the major component of floral emissions of the variety Sonata by Klatt et al. (2013) and Ceuppens et al. (2015), respectively. In our study, these compounds were present at lower amounts in Sonata flowers. The reason for the quanti- tative differences in these studies is unknown.
In our study,α-muurolene was the major component in volatiles from flowers ofF. vesca. 1,4-Dimethoxybenzene was reported as the major constituent ofF. vescafloral volatile
emissions by Wibe et al. (2014), contributing 96.6% of the total amount. However, neither we nor Blažytė-Čereškienė et al. (2017) detected this compound in the blends released byF. vescaflowers. 1,4-Dimethoxybenzene was one of the major components present in samples ofF. viridisflowers (Blažytė-Čereškienėet al.2017), suggesting that Wibe et al.
(2014) worked with other strawberry species or hybrids, rather thanF. vesca.
The SPME data were used to carry out MDS analyses, which separated volatile blends released fromF. x ananassa andF. vescaflowers. The analyses also explained differences betweenF. vescagenotypes, and partly explained the varia- tion among the volatile blends from the three varieties ofF. x ananassavarieties. Flowers of variety Sonata had the most distinct odor blend.
(c)
90 70 50 30 10 10 30 50 70
& §
& ‡
& † n = 16
n = 12 n = 10
P = 0.212 P = 0.084 P = 0.05
Mean number of the records in each arm (%) (e)
(d)
80 60 40 20 0 20 40 60 80
& PDB
n = 11 P = 0.033
& αM
n = 14 P = 0.583
n = 13 & MS P = 0.944
n = 10 & BO P = 0.169
n = 11 & BA P = 0.248
& FV
n = 12 P = 0.006
n = 11 & FAS P = 0.008
Mean number of the records in each arm (%)
n = 6 & LI P = 0.245
n = 7 & DA P = 0.507
70 50 30 10 10 30 50 70
n = 11 P = 0.959
Arm zone
Air uptake Air supply Air supply
No response zone
Mean number of the records in each arm (%) (a)
(b)
Air Air
Mean number of the records in each arm (%)
70 50 30 10 10 30 50 70
n = 11 P = 0.965
n = 14 P = 0.041
n = 14 P = 0.005
n = 16 P = 0.048
flowering F x ananassa var. Sonata plant non-flowering F x ananassa var. Sonata plant flowering F x vesca plant
non-flowering F x vesca plant
Fig. 5 aSchematic of two-choice olfactometer. Behavioral responses of Anthonomus rubiweevils in two-choice olfactometer to (b) air,codors of flowering and non-floweringFragaria x ananassavariety Sonata and F. vescaplants;dmixtures or single synthetic compounds, found in floral odors ofF. x a.var. Sonata,F. vescaandFragaria viridis;ethree doses of 4-methoxybenzaldehyde. Vertical bars are SEM; n = number of weevils tested; FAS is the six-component blend of 4-methoxybenzaldehyde,
benzaldehyde (BA), benzyl alcohol (BO), methyl salicylate (MS), limo- nene (LI) and decanal (DA); FV is the six-component blend of α- m u u r o l e n e (αM ) , B A , B O , M S , L I a n d D A ; P D B = 1 , 4 - dimethoxybenzene. §,‡ and†represent 4-methoxy-benzaldehyde at doses of 10, 100 and 1000 ng, respectively; Data were analyzed by non- parametric Wilcoxon matched-pairs signed-ranks test
Single cell recordings had previously revealed 58 identified and a few unidentified compounds that elicited responses of SBW olfactory receptors (Bichão et al.2005a,b). We detected 16 of these compounds in strawberry floral volatile emissions.
Despite the differences in composition of blends of vola- tiles from the flowers of different species and varieties of strawberry, there were at least 13 common compounds. Five of the most abundant were benzaldehyde, benzyl alcohol, methyl salicylate, limonene and decanal. These were com- bined with 4-methoxybenzaldehyde andα-muurolene to give blends representative ofF. x ananassa(FAS) andF. vesca (FV), respectively, for testing in bioassays. All these com- pounds elicited electrophysiological responses from antennae of SBW (Bichão et al.2005a).
Olfactory Preferences ofAnthonomus rubi
Our data showed that SBW preferred odors of flowering strawberry over those of non-flowering plants. This was somewhat surprising, as SBW females oviposit in flower buds prior to opening. Possibly, floral volatiles are detected from the bud before opening, or weevils are attracted to the area by neighboring flowers which have already opened. A similar preference for odors released from flowering over non- flowering hosts was also reported for cranberry weevils, Anthonomus musculusSay (Szendrei et al.2009), which have oviposition and feeding strategies similar to SBW. However, only femaleA. musculusshowed preference to odor blends released by blueberry flowers over those of flower buds (Szendrei et al.2009). In our study, we did not detect any sex differences in preference.
Addition of a six-component blends of chemicals to non- flowering plants of both cultivatedF. x ananassavar. Sonata and wild speciesF. vesca, increased attractiveness to SBW, relative to non-flowering plants alone. The FAS blend mim- icking the blend from cultivated strawberry included 4- methoxybenzaldehyde as the major component, while the ma- jor component in the FV blend mimicking wild strawberry wasα-muurolene. The other five components in these two synthetic blends were the same, consistent with the observa- tion that SBW shows no preference for flowering plants of either species.
However, none of the compounds in the two six- component blends increased the preference of SBW when tested as a single compound added to non-flowering plants.
This indicates a synergistic action of the volatiles, a common phenomenon in insect behavioral responses to host plant vol- atiles (Bruce and Pickett2011; Richards et al.2016; Sarkar et al.2017). Olfactory synergism between floral volatiles is less frequently reported compared to odors of vegetative plant parts, possibly due to the activity of individual floral compo- nents rarely having been examined (Metcalf et al. 1995;
Richards et al.2016).
4-Methoxybenzaldehyde, the major component of floral volatiles fromF. x ananassa, actually reduced the attractive- ness of non-flowering plants when added at the highest dose of 1000 ng. A similar phenomenon was reported by Webster et al. (2010), showing that individual components of attractive blends of host-plant volatiles can have repellent activity when presented at higher than natural doses and outside the context of the natural host blend.
1,4-Dimethoxybenzene increased the attractiveness to SBW of non-floweringF. vescaplants when added as a single compound. This compound was previously identified as a major component of floral volatiles of F. viridis (Blažytė- Čereškienėet al.2017; Wibe et al.2014) and increased the attractiveness of SBW aggregation pheromone in field trials (Baroffio et al.2018; Wibe et al.2014). This provides encour- agement that our bioassay results are relevant to the field. We plan to test the active blends from the bioassays for attractive- ness to SBW and/or synergism of the aggregation pheromone in field trials.
Acknowledgements The research has been funded from the project
“Softpest Multitrap”provided by the CORE Organic II Funding Bodies, being partners of the FP7 ERA-Net project, CORE Organic II (Coordination of European Transnational research in Organic Food and Farming systems, project no. 249667). We thank Sara Bruun and Birgitta Svensson for collecting the strawberry blossom weevils and Professor Johan A. Stenberg at Swedish University of Agricultural Sciences, Alnarp, Sweden for access to the collection ofF. vescagenotypes.
Funding Open access funding provided by Stockholm University.
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